Human natural killer (NK) lymphocytes of normal donors kill tumor cells in vitro. The applicant has proposed that proteinases have a crucial role in initiation of this cell-mediated cytotoxicity and had demonstrated that both macromolecular plasma antiproteinases and low m. wt. proteinase substrates and inhibitors block NK. To determine the role(s) of proteinases in NK, plasma will be fractionated for particular antiproteinases that best inhibit NK. These antiproteinases will be used as reagents to couple covalently, "trap" and recover the extracellular or cell surface proteinases required for NK. Low m. wt. substrates and competitive inhibitors will also be used, to define the substrate specificities of proteinases essential for lytic activity. 3H-diisopropylfluorophosphate will be used to radiolabel cellular proteinases and examine if proteinases unique to cellular cytotoxicity can be identified in active form during natural cytotoxicity. It is the ultimate long range goal of this work to define not only the proteinases involved in NK, but also their natural substrates, and how cleavage of these substrates relates to the ultimate death of the tumor cell.